U.S. patent number 9,663,779 [Application Number 13/141,885] was granted by the patent office on 2017-05-30 for nucleic acid purification method.
This patent grant is currently assigned to QIAGEN GMBH. The grantee listed for this patent is Roland Fabis, Jan Petzel, Sabine Scheltinga. Invention is credited to Roland Fabis, Jan Petzel, Sabine Scheltinga.
United States Patent |
9,663,779 |
Fabis , et al. |
May 30, 2017 |
Nucleic acid purification method
Abstract
The present invention relates to a method for purifying nucleic
acids from a sample containing nucleic acids, the method comprising
at least the following steps: a. bringing the sample containing
nucleic acids into contact with a nucleic acid binding phase
comprising protonatable groups, wherein the protonatable groups
have a pKs value of 9 to 12; b. binding the nucleic acids to the
nucleic acid phase at a pH (binding pH) that is at least one pH
unit less than the pKs value of at least one of the protonatable
groups; c. eluting the nucleic acids at a pH greater than the
binding pH but at least one pH unit less than the pKs value of at
least one of the protonatable groups. Also disclosed are
corresponding kits and nucleic acid binding phases that can be used
for purifying nucleic acids.
Inventors: |
Fabis; Roland (Leverkusen,
DE), Petzel; Jan (Solingen, DE),
Scheltinga; Sabine (Krefeld, DE) |
Applicant: |
Name |
City |
State |
Country |
Type |
Fabis; Roland
Petzel; Jan
Scheltinga; Sabine |
Leverkusen
Solingen
Krefeld |
N/A
N/A
N/A |
DE
DE
DE |
|
|
Assignee: |
QIAGEN GMBH (Hilden,
DE)
|
Family
ID: |
41698043 |
Appl.
No.: |
13/141,885 |
Filed: |
December 23, 2009 |
PCT
Filed: |
December 23, 2009 |
PCT No.: |
PCT/EP2009/067878 |
371(c)(1),(2),(4) Date: |
September 12, 2011 |
PCT
Pub. No.: |
WO2010/072821 |
PCT
Pub. Date: |
July 01, 2010 |
Prior Publication Data
|
|
|
|
Document
Identifier |
Publication Date |
|
US 20120245337 A1 |
Sep 27, 2012 |
|
Foreign Application Priority Data
|
|
|
|
|
Dec 23, 2008 [DE] |
|
|
10 2008 063 003 |
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N
15/101 (20130101) |
Current International
Class: |
C07H
21/00 (20060101); C12N 15/10 (20060101) |
Field of
Search: |
;536/25.4,25.41,25.42 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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2 362 979 |
|
Aug 2000 |
|
CA |
|
10-502052 |
|
Oct 1995 |
|
JP |
|
9-157282 |
|
Jun 1997 |
|
JP |
|
2002-537306 |
|
Nov 2002 |
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JP |
|
2002-543979 |
|
Dec 2002 |
|
JP |
|
2003-104996 |
|
Apr 2003 |
|
JP |
|
2004-501054 |
|
Jan 2004 |
|
JP |
|
2004-521881 |
|
Jul 2004 |
|
JP |
|
2005-520547 |
|
Jul 2005 |
|
JP |
|
WO 95/27718 |
|
Oct 1995 |
|
WO |
|
WO 99/29703 |
|
Jun 1999 |
|
WO |
|
WO 00/69872 |
|
Nov 2000 |
|
WO |
|
WO 02/48164 |
|
Jun 2002 |
|
WO |
|
WO 03/080834 |
|
Jan 2003 |
|
WO |
|
WO2007/065933 |
|
Jun 2007 |
|
WO |
|
WO2008/097342 |
|
Aug 2008 |
|
WO |
|
Other References
Frassineti et al. Nuclear magnetic resonance as a tool for
determining protonation constants of natural polyprotic bases in
solution. Anal Biochem 1995;231:374-82. cited by examiner .
Porath and Axen. Immobilization of Enzymes to Agar, Agarose, and
Sephadex Supports. Methods in Enzymology 1976;44:19-45. cited by
examiner .
Blackman. The coordination chemistry of tripodal tetraamine
ligands. Polyhedron 2005;24:1-39. cited by examiner .
Anonymous: "QIAGEN Purification Technolgies", Oct. 15, 2005,
XP002570999, Retrieved from the Internet:
URL:http://wwwl.qiagen.com/reseurces/info/qiagen.sub.--Purification.sub.--
-technologies.sub.--1.aspx, retrieved on Mar. 2, 2010. cited by
applicant .
Joachimiak, et al., "Application of spermine-Sepharose column
chromatography to the separation of plant-specific transfer
ribonucleic acids and aminoacyl-tRNA synthetases" Journal of
Chromatography, vol. 180, No. 1, Nov. 28, 1979, pp. 157-162. cited
by applicant.
|
Primary Examiner: Riley; Jezia
Attorney, Agent or Firm: Seyfarth Shaw LLP
Claims
The invention claimed is:
1. A method of purifying nucleic acids from a nucleic
acid-containing sample, the method comprising: (a) contacting the
nucleic acid-containing sample with a nucleic acid-binding phase
comprising nucleic acid-binding groups covalently bound to a
support, the nucleic acid-binding groups having one or more
protonatable groups having a pKa from 9 to 12; (b) binding the
nucleic acids to the nucleic acid-binding phase at a binding pH
which is more than one pH unit below the pKa of at least one of the
one or more protonatable groups; and (c) eluting the nucleic acids
at an eluting pH which is above the binding pH, but one, or more
than one, pH unit below the pKa of at least one of the one or more
protonatable groups, wherein the nucleic acid-binding phase further
comprises: (i) diluting groups, wherein the proportion of nucleic
acid-binding groups relative to the diluting groups is <50%; and
(ii) functional groups which are bound to the support and/or the
nucleic acid-binding groups, and which functional groups have a
negative charge during the eluting and promote the release of the
nucleic acids at the eluting pH.
2. The method according to claim 1 wherein the functional groups
are cation exchangers.
3. The method according to claim 1, wherein the binding comprises
one or more of the following: (a) binding at a pH from 3 to 8, from
4 to 7.5, from 4.5 to 7, from 5.5 to 7, or from 6.5 to 7; and/or
(b) binding at a salt concentration of less than 1 M.
4. The method according to claim 1, wherein the eluting comprises
one or more of the following: (a) eluting at a pH of from 7.5 to
10, 8 to 9, or 8.2 to 8.8; and/or (b) eluting at a salt
concentration of less than 1 M; and/or (c) eluting in a solution
selected from the group consisting of water, biological buffers and
organic buffers.
5. The method according to claim 1, further comprising washing the
nucleic acid binding phase with a washing solution after the
binding and before the elution of the nucleic acids.
6. The method according to claim 5 wherein the washing solution is
water or an aqueous solution, the aqueous solution having a salt
concentration of less than 400 mM.
7. The method according to claim 1, wherein the one or more
protonatable groups comprise amino groups.
8. The method of claim 2, wherein the functional groups are acidic
groups.
9. The method of claim 8, wherein the acidic groups are carboxyl
groups.
10. The method of claim 7, wherein the one or more protonatable
groups comprise from one to ten, two to eight, or two to six amino
groups per protonatable group.
11. The method of claim 7, wherein the amino groups have a pKa of
from 10 to 12.
12. The method of claim 7, wherein the amino groups are not
conjugated to one or more electron density-reducing groups.
13. The method of claim 1, wherein the support is an organic
polymer, hydrogel, or inorganic material.
14. The method of claim 13, wherein the support is an organic
polymer selected from the group consisting of polystyrenes,
polyacrylates, polymethacrylates, polyurethanes, nylon,
polyethylene, polypropylene, polysaccharides, polybutylene and
copolymers thereof.
15. The method of claim 13, wherein the support is a hydrogel
selected from the group consisting of agarose, cellulose, dextran,
cross-linked dextran gel, cross-linked allyl dextran N,N'-methylene
bisacrylamide, and chitosan.
16. The method of claim 13, wherein the support is an inorganic
material selected from the group consisting of silica gels, silica
particles, glass, metal oxides, semi-metal oxides, boron oxide,
magnetic particles and substrates with a metal surface.
17. The method of claim 16, wherein the inorganic material is a
substrate with a gold surface.
18. The method of claim 1, wherein the nucleic acid-binding groups
comprise primary and secondary amines.
19. The method of claim 18, wherein the nucleic acid-binding groups
are spermine and/or spermidine.
20. The method of claim 1, wherein the one or more protonatable
groups have, or are, ion exchangers.
21. The method according to claim 3, wherein the binding occurs at
a salt concentration of 0.5 M or less, 0.25 M or less, or 0.1 M or
less.
22. The method according to claim 4, wherein the eluting occurs at
a salt concentration of 0.5 M or less, 0.25 M or less, 0.1 M or
less, 50 mM or less, 0.25 mM or less, 0.15 mM or less, or 10 mM or
less.
23. The method according to claim 5 wherein the washing solution is
water or an aqueous solution, the aqueous solution having a salt
concentration of 200 mM or less, 100 mM or less, 50 mM or less, or
25 mM or less.
24. The method according to claim 1, wherein the proportion of
nucleic acid-binding groups relative to the diluting groups is
.ltoreq.25%.
25. The method according to claim 1, wherein the proportion of
nucleic acid-binding groups relative to the diluting groups is
.ltoreq.15%.
Description
This application is a National Stage of PCT/EP2009/067878, filed
Dec. 23, 2009 which claims priority to German Application No. 10
2008 063 003.9, filed Dec. 23, 2008, the disclosures of which are
incorporated herein by reference in their entirety.
The present invention relates to a method of and a kit for
purifying nucleic acids from a nucleic acid-containing sample.
Various methods of purifying and isolating nucleic acids have been
disclosed in the prior art. These include the use of phenol
chloroform, salting-out methods, the use of ion exchangers and
silica particles.
A known method of nucleic acid purification is the "charge-switch
method". This involves contacting a nucleic acid-binding phase with
a nucleic acid-containing sample at a first pH at which the nucleic
acid-binding phase has a positive charge. This favours binding of
the negatively charged nucleic acids to said phase. The nucleic
acids are released/eluted by adjusting, according to the
charge-switch principle, a second pH which is higher than the pKa
of the nucleic acid-binding phase, in order to invert, or
neutralize, the positive charge. This promotes detachment of the
bound nucleic acids from the nucleic acid-binding phase.
The prior art has disclosed both soluble phases (see, for example,
EP 0 707 077) and solid phases (see, for example, WO 99/29703).
Various solutions are employed for elution, for example solutions
having a very high pH or else biological buffers, in particular
low-salt buffers such as, for example, Tris buffers, in order to
enable the purified nucleic acids to be further processed
immediately, for example in an amplification reaction or a
restriction digestion.
Even when the known methods of purifying nucleic acids are
suitable, there is a need for improving the existing methods, in
particular for purifying the nucleic acids in a particularly gentle
manner.
It is therefore an object of the present invention to improve the
existing methods of purifying nucleic acids.
This object is achieved according to the present invention by a
method of purifying nucleic acids from a nucleic acid-containing
sample, which has at least the following steps: a. contacting the
nucleic acid-containing sample with a nucleic acid-binding phase
having nucleic acid-binding groups, said nucleic acid-binding
groups having at least one protonatable group having a pKa of from
9 to 12; b. binding the nucleic acids to the nucleic acid-binding
phase at a pH (binding pH) which is at least one pH unit below the
pKa of at least one of the protonatable groups; c. eluting the
nucleic acids at a pH which is above the binding pH but at least
one pH unit below the pKa of at least one of the protonatable
groups (elution pH).
The present invention relates to the purification of nucleic acids
by means of a nucleic acid-binding phase which correspondingly has
nucleic acid-binding groups. Such a nucleic acid-binding group has
at least one protonatable group whose pKa is from 9 to 12. The
nucleic acids are bound at a pH below the pKa of at least one of
these protonatable groups. The protonatable groups take up a proton
and, as a result, become positively charged, causing the nucleic
acid-binding phase to bind the negatively charged nucleic acids.
The elution is carried out at a higher pH, thereby reducing the
positive charge of the nucleic acid-binding phase. According to the
invention, however, the elution pH is below the pKa of the
protonatable groups, in particular at least one pH unit below,
preferably at least two pH units below. This has the considerable
advantage of enabling the elution to be carried out also under
gentle conditions. In contrast to the prior art, the present
invention therefore allows elution at a pH which is below the pKa
of the protonatable groups.
According to one embodiment of the present invention, the nucleic
acids are bound at a pH of from 3 to 8. This information relates to
the pH during binding and therefore in the sample. Depending on the
design of the solid phase, the method according to the invention
can also be carried out at very gentle conditions, thus enabling
the nucleic acids to be bound even at a pH of from 4 to 7.5,
preferably from 5 to 7.5, particularly preferably from 5 to 7, and
very particularly preferably from 6.5 to 7, and therefore in the
virtually neutral range. Owing to the fact that the protonatable
groups of the nucleic acid-binding phase have a pKa of from 9 to
12, said groups have even at relatively neutral pH values a
positive charge which is sufficient for allowing effective
attachment of the nucleic acids. Binding can therefore be carried
out under very gentle conditions, preventing the nucleic acids from
damage.
In addition, it has proved to be advantageous to perform binding at
a low salt concentration. According to one embodiment, the salt
concentration is therefore less than 1 M during binding of the
nucleic acids to the nucleic acid-binding phase. Preferably, the
salt concentration is less than 0.5 M, less than 0.25 M or even
less than 0.1 M. A low salt concentration is preferred in order to
optimize binding of the nucleic acids to the solid phase. Ion
concentrations which are too high have an adverse influence on the
ionic interactions of nucleic acid and the nucleic acid-binding
phase. We have found that the binding buffer may also contain
certain amounts of organic substances such as, for example,
carbohydrates, alcohols such as ethanol, methanol, for example, or
acetone and acetonitride. These substances do not impair
binding.
Another important step of the present method is elution of the
nucleic acids. As illustrated, the nucleic acids are released at a
pH which is above the binding pH. Consequently, the protonatable
groups have a smaller positive charge during elution, and this
favours the release of the nucleic acids. In addition, the pH
during elution is at least one pH unit below the pKa of at least
one of the protonatable groups of the nucleic acid-binding phase.
As a result of this, as illustrated above, the elution can be
carried out under particularly gentle conditions.
Depending on the nucleic acid-binding group or nucleic acid-binding
phase employed, the elution is preferably carried out at a pH of
from 7.5 to 11, from 7.5 to 10, preferably at a pH of from 8 to 9
or 8.2 to 8.8. These low pH values achieve particularly
advantageous results because the nucleic acids are released in a
particularly gentle way. Further measures which enable the nucleic
acids to be released at a low pH in a particularly efficient manner
are described below.
In order to enable the isolated nucleic acids to be further
processed immediately in the elution buffer, the latter preferably
has a low salt concentration. According to one embodiment, the salt
concentration is therefore less than 1 M, preferably less than 0.5
M, less than 0.25 M, less than 0.1 M, particularly preferably less
than 50 mM, less than 25 mM or even less than 10 mM. Suitable salts
may be chlorides of alkali metals and alkaline earth metals or
ammonium, other salts of mineral acids, acetates, borates, and
compounds such as Tris, Bis-Tris, and organic buffers such as, for
example, MES, CHAPS, HEPES, and the like. In addition, substances
suitable for elution have been disclosed in the prior art.
To facilitate purification, preferably at least one washing step is
carried out after binding and prior to elution of the nucleic
acids. Preference is given to using aqueous solutions with low salt
concentrations but also water for washing. Preference is given to
salts present in the washing buffers being at a concentration of
less than 400 mM, particularly preferably less than 200 mM, 100 mM,
50 mM and/or even less than 25 mM. The washing buffer may contain
organic components, for example alcohols, polyols, polyethylene
glycols, acetone, acetonitride or carbohydrates. However, the
washing buffers may be without interfering amounts of the
corresponding organic components, so as not to impair subsequent
applications such as, for example, enzymic processing,
amplification reactions and the like ("downstream"
applications).
The nucleic acid-binding phase to be employed according to the
invention may be solid or soluble. Soluble nucleic acid-binding
phases usually precipitate nucleic acids at the binding pH and
release the bound nucleic acids from the precipitate again at the
elution pH. Soluble nucleic acid-binding phases or polymers are
described in the prior art, for example in EP 0 707 077.
According to the preferred embodiment, the nucleic acid-binding
phase is a solid phase. For preparation, the protonatable groups
may be bound, for example, to a solid support material. Details
will be described hereinbelow. Using a solid phase facilitates
removal of the bound nucleic acids from the sample. According to
one embodiment, binding of the nucleic acids is therefore followed
by removal of the solid phase.
According to one embodiment, the protonatable groups are or have
ion exchangers, preferably anion exchangers. Preferred protonatable
groups proven for binding of nucleic acids are amino groups, with
preference being given to primary and secondary amino groups. The
amino groups preferably have a pKa of from 9 to 12, preferably 10
to 12. The nucleic acid-binding group preferably has from 1 to 10,
particularly preferably 2 to 8 and in particular 2 to 6, amino
groups. Examples of preferred nucleic acid-binding groups are
primary, secondary and tertiary mono- and polyamines. These may be
substituted or unsubstituted. Examples are in particular amines of
the formulae R.sub.1R.sub.2R.sub.3N,
R.sub.1R.sub.2N(CH.sub.2).sub.nNR.sub.3R.sub.4
R.sub.1R.sub.2N(CH.sub.2).sub.nNR.sub.3(CH.sub.2).sub.mNR.sub.4R.sub.5
R.sub.1R.sub.2N(CH.sub.2).sub.nNR.sub.3(CH.sub.2).sub.mNR.sub.4(CH.sub.2)-
.sub.oNR.sub.5R.sub.6
R.sub.1R.sub.2N(CH.sub.2).sub.nNR.sub.3(CH.sub.2).sub.mNR.sub.4(CH.sub.2)-
.sub.oNR.sub.5(CH.sub.2).sub.pNR.sub.6R.sub.7
R.sub.1R.sub.2N(CH.sub.2).sub.nNR.sub.3(CH.sub.2).sub.mNR.sub.4(CH.sub.2)-
.sub.oNR.sub.6(CH.sub.2).sub.pNR.sub.6(CH.sub.2).sub.qNR.sub.7R.sub.8
R.sub.1R.sub.2N(CH.sub.2).sub.nNR.sub.3(CH.sub.2).sub.mNR.sub.4(CH.sub.2)-
.sub.oNR.sub.5(CH.sub.2).sub.pNR.sub.6(CH.sub.2).sub.qNR.sub.7(CH.sub.2).s-
ub.rNR.sub.8R.sub.9
R.sub.1R.sub.2N(CH.sub.2).sub.nNR.sub.3(CH.sub.2).sub.mNR.sub.4(CH.sub.2)-
.sub.oNR.sub.5(CH.sub.2).sub.pNR.sub.6(CH.sub.2).sub.qR.sub.7(CH.sub.2).su-
b.rNR.sub.8(CH.sub.2).sub.sNR.sub.9R.sub.10
where
n, m, o, p, q, r and s are, independently of one another, 2 to
8;
R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, R.sub.7,
R.sub.8, R.sub.9 and R.sub.10 are identical or different and are
selected from the group consisting of H, alkyl (branched or
unbranched) and aryl.
Preferred nucleic acid-binding groups are particularly
N-propyl-1,3-propanediamine and pentaethylenehexamine and very
particularly spermine and spermidine.
In addition, it is also possible to use cyclic amines, aromatic
amines or amino-functionalized heterocycles. The amines may bear
substituents, for example alkyl, alkenyl, alkynyl or aromatic
substituents, and additionally the hydrocarbon chains may also be
closed into a ring. The hydrocarbon chains may also have
heteroatoms, such as oxygen, nitrogen, sulphur or silicon, or
branchings.
Other suitable nucleic acid-binding groups are
polyoxyalkyleneamines having one, two of three amino groups. These
are available, for example, under the name "Jeffamine"
polyoxyalkyleneamines. Jeffamines contain primary amino groups
which are bound to the terminals of the polyether backbone. The
polyether backbone may be based on propylene oxide, ethylene oxide
or mixtures thereof; the use of other backbone segments is also
conceivable.
As stated, the amino groups of the amines have pKa values of from 9
to 12, preferably from 10 to 12.
According to the invention, it is also possible to use mixtures of
the corresponding nucleic acid-binding groups or to apply them on a
support.
The nucleic acid-binding groups such as, for example, the amines
may be bound to the support covalently or by electrostatic, polar
or hydrophobic interaction. Preference is given to linking them in
such a way that one (e.g. N-propyl-1,3-propanediamine) to ten,
preferably two to eight, particularly preferably two to six, amino
groups are present by way of a protonatable group per attached
group.
Preferably, the amino groups of the nucleic acid-binding groups are
not conjugated to an electron density-reducing group such as, for
example, a carboxyl group, a carbonyl group, a group with C--C
double bonds or a .beta.-hydroxyethyl group and, as a result, their
pKa is between 9 and 12. Conjugation to an electron
density-reducing group is regarded to be present, if an amino
function and the corresponding, electron density-reducing group are
connected via only three, two or less carbon atoms.
According to a preferred embodiment, the nucleic acid-binding
groups are bound to a support for a solid nucleic acid-binding
phase to be used for nucleic acid purification. Examples of
suitable supports for the nucleic acid-binding groups are organic
polymers such as polystyrene and its derivatives, polyacrylates and
polymethacrylates, and their derivatives, or polyurethanes, nylon,
polyethylene, polypropylene, polybutylene, and copolymers of thebe
materials. In addition, these nucleic acid-binding groups may also
be linked to polysaccharides, in particular hydrogels such as
agarose, cellulose, dextran, cross-linked dextran gel sold under
the trademark SEPHADEX.RTM., allyl dextran and
N,N'-methylenebisacrylamide matrix sold under the trademark
SEPHACRYL.RTM., or chitosan, Furthermore, the nucleic acid-binding
groups may also be attached to inorganic supports such as, for
example, silica gel, glass or other metal oxides and semi-metal
oxides, silica, boron oxide or metal surfaces such as, for example,
gold. Magnetic particles are particularly advantageous with regard
to handling. The nucleic acid-binding groups may be bound to said
supports directly or else via "spacers". They may also be part of a
larger molecule, Examples of spacers are hydrocarbon chains,
polyethylene glycols or polypropylene glycols, and functionalized
silanes, Said spacers may be branched or unbranched.
Chemical functionalities which may be employed for attaching the
nucleic acid-binding groups are acid amides or acid anhydrides,
epoxides, tresyl groups, formyl groups, sulphonyl chlorides,
maleimides or carbodiimide chemistry-activated carboxylate groups.
It is likewise possible within the scope of the invention to attach
the nucleic acid-binding groups such as, for example, amines
non-covalently, for example by ionic interactions or by absorptive
processes. The nucleic acid-binding groups may also be attached via
thiols to gold surfaces, for example. Preference is given to
attaching the nucleic acid-binding groups to carboxylated
surfaces.
Other embodiments of the support materials comprise non-magnetic
and magnetic particles, column materials, membranes, and surface
coatings. Mention may also be made of functionalized supports such
as tubes, membranes, non-wovens, paper, reaction vessels such as
PCR vessels, "Eppendorf tubes", multiplates, chips and
microarrays.
Another embodiment of the present invention relates, as stated, to
a soluble polymer which has protonatable groups in accordance with
the present invention and which is capable of reversibly binding
nucleic acids according to the principle according to the
invention. Examples of suitable soluble phases which may also be
modified according to the invention are described, for example, in
EP 0 707 077. The preferred solvent is water but it is also
possible to employ polymers which have been functionalized
according to the invention and which are soluble in organic
solvents such as ethanol, for example.
Surprisingly, we have found that the applicable pH values and salt
concentrations in the binding and elution buffers correlate with
the number of protonatable groups, in particular amino groups,
present per nucleic acid-binding group. Thus, at a salt
concentration of approx. 50 mM, the nucleic acid binds to
spermine-coated surfaces even at pH 6, while a lower pH of 5.5,
preferably even 5, is preferred for application of an
N-propyl-1,3-propanediamine-coated surface. Elution from
N-propyl-1,3-propanediamine surfaces is successful even at pH 7.5,
while a pH of approx. 8.5 is required with spermine-coated surfaces
at a salt concentration of 50 mM. However, the pH may also still be
lowered for elution by modifying the support (see below).
An efficient elution and consequently detachment of the bound
nucleic acids from the nucleic acid-binding phase is particularly
important for the efficiency of nucleic acid purification. Here, it
was surprisingly found that it is not only the pKa values of the
protonatable groups of the nucleic acid-binding groups that are
important. The structure of the nucleic acid-binding phase and the
presence of other functional groups also contribute to facilitating
and improving elution at pH values in the neutral or weakly
alkaline range.
According to one embodiment, the nucleic acid-binding phase
additionally bears functional groups which promote elution of the
nucleic acids at the elution pH, for example by exerting a
repelling effect. These functional groups therefore preferably have
a negative charge during elution. The pKa values of these groups
may be, for example, in a range from 0 to 7, preferably 1 to 5.
Suitable are, for example, ion exchangers, in particular cation
exchangers, preferably acidic groups such as, for example, carboxyl
groups. Other suitable groups are betaines, sulphonates,
phosphonates and phosphates. For example, the solid support may be
functionalized with carboxyl groups to enable the nucleic
acid-binding groups to be attached. The concentration of the
nucleic acid-binding groups for attachment will be chosen in such a
way that some of the carboxyl groups are free and therefore not
functionalized with the nucleic acid-binding groups. These groups
do not impair binding of the nucleic acids at low pH values. At
higher pH values, however, they are preferably negatively charged
and, as a result, facilitate detachment of the nucleic acids from
the nucleic acid-binding groups. This interaction may also be
facilitated by selection of the length or the distance between the
protonatable groups of the nucleic acid-binding groups and the
negative-ionizable groups such as, for example, the carboxyl
groups. This advantageously facilitates elution at low pH values,
thus increasing the yield. The selection, strength and length of
the functional groups exerting a repelling effect on the nucleic
acids at the elution pH vary, depending on the nucleic acid-binding
group selected, thus in particular the number of protonatable
groups per nucleic acid-binding group and their distance to the
elution-promoting functional groups.
We have furthermore demonstrated that the elution efficiency can
also be increased if the nucleic acid-binding/protonatable groups
are arranged at a distance to one another on the support material
or diluted. According to one embodiment, such an arrangement of the
nucleic acid-binding groups can be achieved by coating the support
only with small amounts of nucleic acid-binding groups. Preference
is therefore given to carrying out functionalization with a
substoichiometric amount of nucleic acid-binding groups. As a
result, the nucleic acid-binding groups are basically diluted on
the support and, consequently, fewer nucleic acid-binding groups
are available. This facilitates elution because the nucleic acids
bind less tightly and can therefore be detached again more readily
from the nucleic acid-binding phase. For example, only .ltoreq.50%,
.ltoreq.25%, .ltoreq.15%, .ltoreq.10% or only .ltoreq.5% of the
functional groups on the support material may be functionalized
with nucleic acid-binding groups. If the support material does not
have any suitable functional groups for attaching the nucleic
acid-binding groups, the support material may be functionalized
first in order to provide it with suitable functional groups (see
above). To this end, the appropriate profile of coating of the
support material with a substoichiometric amount of nucleic
acid-binding groups may also be achieved by providing the support
material correspondingly with fewer functional groups for attaching
said nucleic acid-binding groups.
According to another embodiment, the support is coated with a
mixture of nucleic acid-binding groups and "diluting groups". The
term "diluting groups" is used herein for illustrating its function
in relation to the nucleic acid-binding groups. Their function
comprises adjusting the amount of nucleic acid-binding groups on
the support and thereby also influencing the strength of binding of
the nucleic acids. The higher the proportion of diluting groups,
the fewer nucleic acid-binding groups are applied to the support
and the lower is the strength of binding to the nucleic acids. The
diluting groups may have a negative, positive or neutral charge or
ionizable groups. Consequently, the diluting groups may
simultaneously also have functional groups which promote elution
(see above). The proportion of nucleic acid-binding groups in
relation to the diluting groups may be, for example, .ltoreq.50%,
.ltoreq.25%, .ltoreq.15%, .ltoreq.10% or only .ltoreq.5%. Examples
of suitable diluting groups are amines, dimethylamine, diethylamine
and ammonia. According to a preferred embodiment, a mixture of
common (known in the prior art) monoamines and polyamines according
to the invention is applied to the support. The polyamines in this
combination are used for attaching the nucleic acids, with the
mono-amines acting primarily as diluting groups. An example of a
suitable diluting group would be ethanolamine.
The pH of the nucleic acid-binding phase may be optimized with
respect to the elution conditions by choosing/combining the
parameters described, in particular the functional groups promoting
elution, the diluting groups, and the dilution or mixture with
nucleic acid-binding groups. Correspondingly, the elution profile
of the nucleic acid-binding phase, in particular the elution pH,
may be controlled or adjusted.
Nucleic acids which may be purified by the systems according to the
invention may he present in body fluids such as blood, urine,
stool, saliva, sputum, or other body fluids, in biological sources
such as tissue, cells, in particular animal cells, human cells,
plant cells, bacterial cells and the like, organs such as liver,
kidneys or lungs, etc. In addition, the nucleic acid may he
obtained from support materials such as swabs, pap smears, and
stabilizing media such as the methanol-water solution sold under
the trademark PRESERVCYT.RTM. or the liquid-based Pap test sold
under the trademark SUREPATH.RTM., or else from other liquids such
as, for example, juices, aqueous samples or food in general. In
addition, the nucleic acids may be obtained from plant material,
bacterial lysates, paraffin-embedded tissue, aqueous solutions or
gels.
In addition, the present invention relates to the use of a nucleic
acid-binding phase as described above for purifying nucleic acids.
The nucleic acid-binding phase employed according to the invention
has in particular nucleic acid-binding groups with at least one
protonatable group having a pKa of from 9 to 12. Preferred
embodiments are described in detail above (see disclosure above)
and are characterized in particular by one or more of the following
features: a) the nucleic acid-binding phase is solid or soluble;
and/or b) the nucleic acid-binding phase has nucleic acid-binding
groups bound to a solid support; and/or c) the nucleic acid-binding
phase additionally has functional groups which promote
release/elution of the nucleic acids at the elution pH, preferably
cation exchangers, in particular carboxyl groups; and/or d) the
nucleic acid-binding phase according to feature b) or c) has a
support selected from the group consisting of organic polymers such
as polystyrene and its derivatives, polyacrylates and
polymethacrylates and their derivatives, polyurethanes, nylon,
polyethylene, polypropylene, polybutylene and copolymers of these
materials, polysaccharides and hydrogels such as agarose,
cellulose, dextran, cross-linked dextran gel sold under the
trademark SEPHADEX.RTM., allyl dextran and
N,N'-methylenebisacrylamide matrix sold under the trademark
SEPHACRYL.RTM., chitosan, inorganic supports, in particular silica
gels, silica particles, glass or other metal and semi-metal oxides,
boron oxide, supports with metal surfaces, for example gold and
magnetic particles; and/or e) the nucleic acid-binding groups of
the nucleic acid-binding phase are amines, in particular primary
and secondary amines; and/or f) the nucleic acid-binding groups
according to feature e) are in particular spermine and/or
spermidine.
The invention further provides a kit for purifying nucleic acids,
which kit is characterized in that it has a) a nucleic acid-binding
phase according to the invention, which has nucleic acid-binding
groups with at least one protonatable group having a pKa of from 9
to 12; b) a binding buffer with a pH which is at least one pH unit
below the pKa of at least one of the protonatable groups of the
nucleic acid-binding phase, and/or a binding buffer which enables
such a pH to be adjusted in the sample; c) an elution buffer with a
pH which is at least one pH unit below the pKa of at least one of
the protonatable groups of the nucleic acid-binding phase but above
the pH of the binding buffer, and/or an elution buffer which
enables such a pH to be adjusted in the sample.
Details of the nucleic acid-binding phase and the elution
conditions are described above and also apply in connection with
the kit according to the invention and characterize the
components/buffers used therein. Reference is made to the
disclosure above. In addition, the kit may contain other customary
components such as, for example, lyses, washing and/or neutralizing
reagents and/or buffers.
The binding buffer may preferably have at least one of the
following features: i. a pH of from 3 to 8; and/or ii. a pH of from
4 to 7.5; and/or iii. a pH of from 4.5 to 7; and/or iv. a pH of
from 5.5 to 7; and/or v. a pH of from 6.5 to 7; and/or vi. a salt
concentration of less than 1 M, less than 0.5 M, less than 0.25 M
or less than 0.1 M.
The advantages of the corresponding features have been illustrated
above in connection with the method, and reference is made to the
disclosure above.
The elution buffer according to the invention may have at least one
of the following features: i. a pH of from 7.5 to 10; and/or ii. a
pH of from 8 to 9; and/or iii. a pH of from 8.2 to 8.8; and/or iv.
a salt concentration of less than 1 M, less than 0.5 M, less than
0.25 M, less than 0.1 M, less than 25 mM, less than 15 mM, or less
than 10 mM; and/or v. it is selected from the group consisting of
water, biological buffers, organic buffers, in particular Tris,
Tris-Bis, MES, CHAPS and HEPES.
Details and advantages of the corresponding features have been
illustrated above in connection with the method according to the
invention. Reference is made to the disclosure above.
The corresponding kits may be applied in particular within the
framework of the method according to the invention. The present
methods, kits and nucleic acid-binding solid phases may be employed
in particular in the field of molecular biology, molecular
diagnostics, in forensics, in food analysis and in applied
testing.
Preference is given to enabling the eluted nucleic acids to be
further processed immediately, thus to be used, for example, in a
PCR, RT-PCR, a restriction digestion or a transcription. Further
purification is not required, as long as the elution buffers are
designed as described above and preferably have a low salt
concentration.
Nucleic acids suitable for purification are DNA and RNA, in
particular genomic DNA, plasmid DNA, and also PCR fragments, cDNA,
miRNA, siRNA, and also oligonucleotides and modified nucleic acids
such as, for example, PMA or LMA. It is also possible to purify
viral or bacterial RNA and DNA or nucleic acids from human, animal
or plant sources. Furthermore suitable for a purification according
to the invention are also DNA/RNA hybrids.
The present invention will be illustrated below on the basis of
some examples. These examples are not limiting but are preferred
embodiments of the present invention. In addition, all references
cited herein are made subject matter of the disclosure.
EXAMPLES
Model systems of nucleic acids that were employed in the
experiments are pUC21 plasmid DNA, uncut, RNA, and genomic DNA. In
addition, the purification of nucleic acid fragments of different
sizes was demonstrated using plasmid DNA cut into fragments by
restriction enzymes.
The procedure (in the experimental part) followed the preparation
protocols A) to I) below:
A) Reaction of Magnetic Polymers with Amines
Materials
Magnetic polymer: Carboxylate-modified magnetic particles sold
under the trademark SERA-MAG.RTM. Double Speed Magnetic
Carboxylate-Modified Microparticles (dsMGCM) catalogue No.
65152105050250, 5% strength aqueous suspension, or Magnetic
Carboxylate-Modified (MG-CM), catalogue No. 2415-2105, 5% strength
aqueous suspension, Seradyn Inc. Indianapolis, USA.
Amines: spermine (Fluka, 85590), spermidine (Fluka, 85561),
propyl-1,3-propanediamine (Aldrich, catalogue No. 308153),
pentaethylenehexamine (Aldrich, catalogue No. 292753)
poly(allylamine hydrochloride), Mw 15 000 (Aldrich, catalogue No.
283215)
500 mg of the magnetic particles are resuspended in 10 ml of 50 mM
MES buffer, pH 6.1, and then admixed with 11.5 ml of a 50 mg/ml
solution of N-hydroxysuccinimide. After mixing using a minishaker,
10 ml of a 52 .mu.mol/l solution of
1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) are added;
followed by another vortexing. The solution is then left to react
on an end-over-end shaker for 30 minutes, and the supernatants are
then removed. After resuspension in 50 ml of 50 mM MES buffer, pH
6.1, the suspension is distributed in 10 ml aliquots. The
suspension is magnetically separated and the supernatants are
discarded. After suspension in 1 ml of 50 mM MES buffer, pH 6.1, in
each case 2 ml of the amine are added at a concentration of 100
mg/ml in 50 mM MES and a pH 8.5, followed by thorough vortexing,
sonication for 10 minutes, and the suspension is left to react on
an end-over-end shaker for one hour. This is followed by washing
twice with in each case 10 ml of 50 mM MES buffer, pH 6.1, magnetic
separation and discarding of the supernatants. The particles are
then resuspended in each case 2 ml of MES buffer at pH values from
4.5 to 7.0.
B) Purification of Plasmid pUC21 Using
N-propyl-1,3-propanediamine-functionalized Magnetic Polymers
(AX027)
2 mg of the magnetic particles in 50 mM MES buffer, pH 5.0 or 5.5
are used and admixed in each case with 50 .mu.l of 50 mM MES
buffer, pH 5.0 or 5.5. This is followed by adding 10 .mu.g of
plasmid pUC21 in 10 .mu.l of buffer "EB" (QIAGEN, catalogue No.
19068) and mixing by way of brief shaking. The reaction mixture is
then incubated on an end-over-end shaker or Eppendorf shaker for 5
minutes. The sample mixture is magnetically separated and the
supernatants are removed and the DNA content is determined
photometrically. The residues are then washed twice with in each
case 100 .mu.l of Millipore water, magnetically separated, and the
supernatants are discarded. This is followed by eluting twice by
adding in each case 50 .mu.l of 50 mM Tris buffer, pH 8.5 with NaCl
concentrations of 50 mM, 100 mM, 200 mM and 400 mM, removal by
means of magnetic separation and examining the eluates
photometrically for their DNA content.
FIG. 1 depicts the results for 50 mM NaCl concentrations, also in
comparison with AX 026 and AX 027.
C) Purification of Plasmid pUC21 Using Spermidine-functionalized
Magnetic Polymers (AX 026)
2 mg of the magnetic particles in 50 mM MES buffer, pH 6.2 are used
and admixed with 50 .mu.l of 50 mM Tris buffer, pH 6.2. This is
followed by adding 10 .mu.g of plasmid pUC21 in 10 .mu.l of buffer
"ES" (QIAGEN, catalogue No. 19068) and mixing by way of brief
shaking. The reaction mixture is then incubated on an end-over-end
shaker or Eppendorf shaker for 5 minutes. The sample mixture is
magnetically separated and the supernatants are removed and the DNA
content is determined photometrically. The residues are then washed
twice with in each case 100 .mu.l of Millipore water, magnetically
separated, and the supernatants are discarded. This is followed by
eluting twice by adding in each case 50 .mu.l of 50 mM Tris buffer,
pH 7.5, 8.0 and 8.5 with NaCl concentrations in each case of 50 mM,
100 mM, 200 mM and 400 mM, removal by means of magnetic separation
and examining the eluates photometrically for their DNA
content.
The results are depicted in FIG. 2.
D) Purification of Plasmid pUC21 Using Spermine-functionalized
Magnetic Polymers (AX 025)
2 mg of the magnetic particles in 50 mM MES buffer, pH 6.1 are used
and admixed with 50 .mu.l of 50 mM Tris buffer, pH 7.0. This is
followed by adding 10 .mu.l of plasmid pUC21 in 10 .mu.l of buffer
"EB" (QIAGEN, catalogue No. 19068) and mixing by way of brief
shaking. The reaction mixture is then incubated on an end-over-end
shaker or Eppendorf shaker for 5 minutes. The sample mixture is
magnetically separated and the supernatants are removed and the DNA
content is determined photometrically. The residues are then washed
twice with in each case 100 .mu.l of Millipore water, magnetically
separated, and the supernatants are discarded. This is followed by
eluting twice by adding in each case 50 .mu.l of 50 mM Tris buffer,
pH 7.5, 8.0 and 8.5 with NaCl concentrations in each case of 50 mM,
100 mM, 200 mM and 400 mM, removal by means of magnetic separation
and examining the eluates photometrically for their DNA
content.
The results are depicted in FIG. 3.
E) Purification of Plasmid pUC21 Using
Pentaethylenehexamine-functionalized Magnetic Polymers (AX 028)
2 mg of the magnetic particles in 50 mM MES buffer, pH 6.1 are used
and admixed with 50 .mu.l of 50 mM Tris buffer, pH 7.0. This is
followed by adding 10 .mu.g of plasmid pUC21 in 10 .mu.l of buffer
"EB" (QIAGEN, catalogue No. 19068) and mixing by way of brief
shaking. The reaction mixture is then incubated on an end-over-end
shaker or Eppendorf shaker for 5 minutes. The sample mixture is
magnetically separated and the supernatants are removed and the DNA
content is determined photometrically. The residues are then washed
twice with in each case 100 .mu.l of Millipore water, magnetically
separated, and the supernatants are discarded. This is followed by
eluting twice by adding in each case 50 .mu.l of 50 mM Tris buffer,
pH 7.5, 8.0 and 8.5 with NaCl concentrations in each case of 50 mM,
100 mM, 200 mM and 400 mM, removal by means of magnetic separation
and examining the eluates photometrically for their DNA
content.
The results are depicted in FIG. 4.
F) Purification of Plasmid pUC21 Using
Polyallylamine-functionalized Magnetic Polymers (AX
029)--Comparative Example
2 mg of the magnetic particles in 50 mM MES buffer, pH 6.1 are used
and admixed with 50 .mu.l of 50 mM Tris buffer, pH 7.0. This is
followed by adding 10 .mu.g of plasmid pUC21 in 10 .mu.l of buffer
"EB" (QIAGEN, catalogue No. 19068) and mixing by way of brief
shaking. The reaction mixture is then incubated on an end-over-end
shaker or Eppendorf shaker for 5 minutes. The sample mixture is
magnetically separated and the supernatants are removed and the DNA
content is determined photometrically. The residues are then washed
twice with in each case 100 .mu.l of Millipore water, magnetically
separated, and the supernatants are discarded. This is followed by
eluting twice by adding in each case 50 .mu.l of 50 mM Tris buffer,
pH 7.5, 8.0 and 8.5 with NaCl concentrations in each case of 50 mM,
100 mM, 200 mM and 400 mM, removal by means of magnetic separation
and examining the eluates photometrically for their DNA
content.
The results are depicted in FIG. 5.
G) Purification of Genomic DNA Using Spermine-functionalized
Magnetic Polymers (AX 030)
For each purification, 2 mg of particles are suspended in 25 mM
MES, 25 mM Tris, pH 6.2. This is followed by adding 10 .mu.g of
calf thymus genomic DNA (catalogue No. 89370, Fluka, Germany) in
buffer "EB" and mixing by way of brief shaking. This is followed by
magnetic separation and photometric examination of the. The residue
is washed with 100 .mu.l of Millipore water with magnetic
separation to remove the supernatants, followed by eluting twice
with in each case 50 .mu.l of 50 mM TRIS buffer and 50 mM and 100
mM NaCl, respectively, pH 8.5. The DNA content of the individual
eluates is then determined photometrically.
The results are depicted in FIG. 6 and FIG. 7.
H) Purification of RNA Using Spermine-functionalized Magnetic
Polymers (AX 030)
For each purification, 2 mg of particles are suspended in 50 mM
Tris buffer, pH 5.5. This is followed by adding 10 .mu.g/prep. RNA
(16S- & 23S ribosomal, Fermentas 41-1 g/l-ll) in 50 mM Tris
buffer, pH 5.5. This is followed by mixing by way of brief shaking,
magnetic separation and the supernatants are then examined
photometrically for RNA. This is followed by washing twice with in
each case 100 .mu.l of RNase-free water and removing the
supernatants by magnetic separation. This is followed by eluting
twice with in each case 50 .mu.l or 50 mM TRIS (RNase-free), pH 8.5
and 50 mM and 100 mM NaCl, respectively. The eluates are then
photometrically examined separately.
The results are depicted in FIG. 8 and FIG. 9.
I) Purification of Nucleic Acid Fragments Using
Spermine-functionalized Magnetic Polymers (AX 030)
Preparation
The spermine-functionalized magnetic polymer particles are
suspended in a binding buffer containing 25 mM MES, 25 mM Tris, pH
6.2, at a suspension density of 50 mg/ml. The beads are then washed
another two times with this buffer, and the supernatants are
removed by means of magnetic separation. Type pTZ19R plasmid DNA is
required, of which 25 .mu.g of DNA are to be used for 100 .mu.l of
buffer solution. First, all quantities for the reaction mixture are
calculated, followed by introducing the missing amount of water to
add up to 100 .mu.l, adding the DNA, then the matching enzyme
buffer for the enzyme solution (1 .mu.l per 10 .mu.l of total
solution), then finally 3U of restriction enzyme (Hinf I, New
England Biolabs, Cat. No. R0155S) per .mu.g of DNA (usually 75 U
correspond to 7.5 .mu.l of enzyme solution). The mixture is left to
incubate in a water bath or heat block at 37.degree. C. for 90
minutes. This is followed by brief centrifugation using Quick-Run
to 6000 U/min, and the samples are then frozen at -20.degree. C.
This restriction-digested DNA is a simple and rapid PCR replacement
for assaying the PB buffers.
Procedure
For each sample, 8 .mu.l of the PCR solution (corresponding to 2
.mu.g of DNA) are admixed with 92 .mu.l of 25 mM MES, 25 mM Tris,
pH 6.2, and then with 25 .mu.l of the magnetic silica gel
suspensions, followed by vortexing for 10 seconds, admixing with
500 .mu.l of 25 mM MES, 25 mM Tris, pH 6.2, and mixing. The mixture
is incubated in a shaker for 2 minutes. This is followed by
magnetic separation, discarding of the supernatant and washing
twice with in each case 750 .mu.l of Millipore water. This is
followed by eluting first with 50 .mu.l, then with 30 .mu.l of 50
mM Tris/HCl, 50 mM NaCl, pH 8.5. The eluates are combined and
examined photometrically, and also, by way of a gel, for DNA
content.
The results are depicted in FIG. 10, FIG. 11 and FIG. 12.
J) Purification of Genomic DNA from Blood Using
Spermine-functionalized Magnetic Polymers (AX 040)
1 ml of lysis buffer (10 mM TRIS, polyethylene glycol
p-(1,1,3,3-tetramethylbutyl)-phenyl ether sold under the trademark
TRITON.TM. X-100, pH 9.0) and 10 .mu.l of proteinase K are mixed. 1
mg of beads and 3.4 .mu.l of water are suspended in 200 .mu.l of
bind buffer (1.5 M potassium acetate pH 4.0) at a suspension
density of 26.6 mg/ml. 100 .mu.l of thawed whole blood
(citrate-stabilized) are admixed with the lysis buffer/proteinase
mix in an Eppendorf cup and mixed well. This is followed by
incubation at room temperature for 10 minutes. The beads are
carefully resuspended in binding buffer. Of this, 240 .mu.l are
added to the lysed blood, followed by careful mixing by pipetting
up and down. This is followed by incubation at room temperature for
1 min. The beads are magnetically separated. The supernatant is
removed with care, For washing, 1 ml of washing buffer (water) is
added, followed by careful mixing by pipetting up and down. After
another magnetic separation, the supernatant is discarded. 1 ml of
lysis buffer and 50 .mu.l of binding buffer are added, mixed
carefully and incubated at RT for 1 min. Magnetic separation is
followed once more by washing with 1 ml of washing buffer and
magnetic separation.
The purified DNA is eluted by adding 150 .mu.l of elution buffer
(10 mM TRIS*HCl pH 8.5) to the beads. The beads are resuspended by
pipetting up and down several times. The beads are magnetically
separated, the supernatant is removed, and the DNA is
photometrically quantified.
The results are depicted in FIG. 13.
5 .mu.l of the eluate obtained above are subjected to RT-PCR. For
this TAQMAN.RTM. .beta.-actin control reagent (Applied Biosystems,
No. 401846) and QUANTITECT.RTM. Probe PCR Mastermix (QIAGEN, No.
1019337) are used, with 12.5 .mu.l of Mastermix, 2.5 .mu.l of
.beta.-actin Probe (6-FAM), 2.5 .mu.l of .beta.-actin Forward
Primer and 2.5 .mu.l of .beta.-actin Reverse Primer being
applied.
The results are depicted in FIG. 14.
* * * * *
References